Mycobacterium tuberculosis (Mtb) infects over 2 billion people worldwide and causes 1.4 million deaths annually. The standard treatment for tuberculosis (TB) due to drug-susceptible Mtb consists of 2 months of rifampin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) followed by 4 months of RIF and INH. In patients with clinical TB, Mtb exists in 3 metabolic states: log phase growth, semi-dormant acidic phase, and a non-replicating persister (NRP) state. NRP Mtb requires prolonged therapy to kill and is responsible for disease relapse. RIF, INH and EMB kill log phase growth Mtb, while PZA kills acidic phase Mtb. RIF also kills NRP Mtb. Thus, only one drug in the standard regimen is active against acidic phase and NRP Mtb. The prevalence of multidrug resistant Mtb (MDR-TB) is rising due to the use of empiric antibiotic combinations for TB caused by microbes that are resistant to one or more drugs in the regimen a priori, errors in the administration of the medications even under Direct Observed Therapy, and patient non-compliance with the long treatment course. In studies in which new antibiotics with novel mechanisms of action are added to the standard regimen for drug-susceptible Mtb and MDR-TB the time to bacterial sterilization in animal models and the time for sputum conversion to negative in clinical trials are shortened, showing that regimens consisting of the standard first and second line TB drugs are not optimized to kill Mtb. Our long term objective is to develop improved TB regimens. The overarching hypothesis is that TB regimens that are pharmacodynamically (PD) optimized to kill Mtb in all 3 metabolic states and to prevent amplification of less-susceptible bacterial subpopulations will provide a potent shorter course TB regimen that will improve treatment outcomes and reduce resistance. We will test this hypothesis and develop a highly effective short course regimen by completing the following Specific Aims: Specific Aim #1. Simulating in an in vitro hollow fiber infection model (HFIM) the free pulmonary PK profiles for clinically relevant doses of 3 novel TB antibiotics that have activity in all metabolic states, identify the P-indices, drug exposures, and dosing intervals of each drug that PD-optimizes the rapidity and extent of killing of DS-Mtb in each of the 3 metabolic states. Determine if these single drug regimens can prevent resistance. Specific Aim #2. With the HFIM, compare the rates and extents of killing of DS-Mtb in the 3 metabolic states and the effect of these antibiotics on the less susceptible Mtb population when the PD-optimized regimens developed in Specific Aim #1 are used as 2 and 3 drug combinations. Employ innovative mathematical models to identify the dose and frequency of administration of each antibiotic in a 3 drug regimen that is predicted to provide a shorter course, highly effective regimen for the treatment of human TB by optimizing the killing of Mtb in each metabolic state and by preventing resistance. Specific Aim #3. Using the HFIM, characterize the efficacy of the PD-optimized 3 drug regimen on the rate and extent of killing of Mtb in 3 metabolic states for strains that are resistant to 1 of the drug components. Specific Aim #4. Prospectively validate the performance of the innovative PD-optimized 3 drug regimen in a novel murine model of pulmonary TB in which Mtb in log phase, acidic phase, and NRP state co-exist and in another innovative in vivo model of TB using state-of-the-art dosing algorithms that humanize the PK profiles generated in the animals. Use the novel murine model to characterize the relative efficacy of this regimen for the killing of DS- and MDR-TB.